1. Field of the Invention
The present invention relates to the fields of molecular biology, gerontology, and medical pharmacology and diagnostics.
2. Description of Related Art
There is substantial evidence that somatic cells have a finite replicative capacity (Hayflick and Moorhead, 1961, Exp. Cell Res. 25: 585-621; Hayflick, 1965, Exp. Cell Res. 37: 614-636; and Hayflick, 1970, Exp. Geront. 5: 291-303) and that this process is a major etiological factor in aging and age-related disease (Goldstein, 1990, Science 249: 1129-1133; Stanulis-Praeger, 1987, Mech. Ageing Dev. 38: 1-48; and Walton, 1982, Mech. Ageing Dev. 19: 217-244). As cells undergo replicative senescence in vitro and in vivo, the cells not only lose the ability to divide in response to growth stimuli, but there are also significant deleterious changes in the pattern of gene expression (West, 1994, Arch. Derm. 130: 87-95). As an individual grows older, senescent cells make up an increasing percentage of the cells present in the tissues of the aging individual. The altered pattern of gene expression exhibited by senescent cells is likely to contribute significantly to age-related pathologies. Reversal of, or a delay in the onset of, senescence should provide an effective therapy for diseases in which replicative senescence plays a role.
There is growing evidence that the fundamental cause of cellular senescence is the progressive loss or telomeric repeated DNA in somatic cells that lack the enzyme designated telomerase (see Harley, 1991, Telomere loss: Mitotic clock or genetic time bomb? Mut. Res. 256:271-282). There is currently no consensus as to the molecular mechanisms that recognize the shortened telomeres in aged cells and cause a cell cycle arrest at the G.sub.1 /S interface, but this arrest may be caused by a DNA checkpoint arrest in which the senescent cell recognizes the shortened telomere as damaged DNA and causes cell cycle arrest similar to that observed in normal cells, which arrest their growth in the presence of DNA damage.
The mammalian enzyme Poly (ADP-Ribose) Polymerase (PADPRP) has been implicated in the signalling of DNA damage. PADPRP activity is higher in isolated nuclei of SV40-transformed fibroblasts than in those of untransformed fibroblasts; leukemic cells show higher enzyme activity than normal leukocytes; and colon cancers show higher enzyme activity than normal colon mucosa (see Miwa et al., 1977, Arch. Biochem. Biophys. 181: 313-321; Burzio et al., 1975, Proc. Soc. Exp. Biol. Med. 149:933-938; and Hirai et al., 1983, Cancer Res. 43:3441-3446. These observations led to the conclusion that the enzyme activity responds to DNA damage and parallels DNA repair. Supporting this conclusion is the observation that the reduction of the activity of the enzyme by certain drugs increases DNA amplification and consequent oncogenesis in cells (see Harris, 1985, Int. J. Radiat. Biol. 48: 675-690).
More recent work has focused on the mechanism by which PADPRP modulates DNA replication and repair (see Smulson and Sugimura, eds., "Novel ADP-ribosylations of regulatory enzymes and proteins," Elsevier, N.Y. (1980)). Such studies have identified PADPRP as an .about.113 kDa protein that uses AND as a substrate in the formation of poly (ADP-ribose) chains at sites on many nuclear proteins. The enzyme binds tightly to DNA and requires DNA strand breaks for activity (see Benjamin and Gill, 1980, J. Biol. Chem. 255: 10502-10508). The PADPRP enzyme system appears to function in response to transient and localized DNA strand breaks in cells that may arise through a variety of processes including DNA repair, replication, recombination, and gene rearrangement (see Alkhatib et al., 1987, Proc. Natl. Acad. Sci. USA 84:1224-1228). The cDNA corresponding to the PADPRP gene has also been cloned and sequenced (see Cherney et al., 1987, Proc. Natl. Acad. Sci. USA 84:8370), and methods for detecting a predisposition to cancer arising out of mutations in the PADPRP gene have been reported (see U.S. Pat. No. 5,272,057).
Inhibitors of PADPRP have also been developed, primarily for the purpose of enzymatic studies (see Banasik et al., 1992, J. Biol. Chem. 267: 1569-1575) and for use in cancer and anti-viral therapies (see PCT patent publication No. 91/18591). PADPRP inhibitors have been reported to be effective in radiosensitizing hypoxic tumor cells (see U.S. Pat. Nos. 5,032,617; 5,215,738; and 5,041,653). These compounds can also be used to prevent tumor cells from recovering from potentially lethal damage of DNA after radiation therapy, presumably by their ability to prevent DNA repair.
One weak inhibitor of PADPRP known as kinetin (Althaus, F. R., and Richter, C., 1987, ADP-ribosylation of Proteins (Springer-Verlag); see p. 26) has also been reported to delay the onset of aging characteristics in human fibroblasts (see Rattan and Clark, 1994, Biochem. Biophys. Res. Comm. 201(2): 665-672). However, the researchers speculated that kinetin acted through receptor-mediated action on the components of protein synthetic machinery, improving the efficiency of various maintenance and repair pathways such as fidelity of protein synthesis, scavenging free radicals, and removing abnormal and damaged macromolecules. Moreover, the researchers stated that the anti-aging effects of kinetin were not accompanied by an increase in cell culture lifespan in terms of maximum proliferative capacity in vitro.
Consequently, there remains a need for compounds that can delay the onset of senescence and extend the maximum proliferative capacity of cells in vivo and in vitro. The present invention meets this and other needs.